title: Laminar shear, but not orbital shear, has a synergistic effect with thrombin stimulation on tissue factor expression in human umbilical vein endothelial cells.
abstract: BACKGROUND
  High levels of tissue factor (TF) have been associated with atherosclerotic plaques. The specific pathways linked to TF expression in endothelial cells (ECs) have not been well defined. This study compared TF expression in human umbilical vein ECs (HUVECs) exposed to laminar shear stress (LSS) using a parallel flow chamber and to orbital shear stress (OSS) using an orbital shaker. We also compared the effects of thrombin (TH) stimulation of ECs exposed to different shear forces on the expression of TF and investigated the role that second messengers, p38 and extracellular signal-regulated kinase 1 and 2 (ERK1/2), had in the EC response.
  
  
  METHODS
  HUVECs were subjected to 2, 4, or 6 hours of LSS or OSS in the presence or absence of 4 U/mL of TH. Western blot analysis of ERK1/2 and p38 activation and polymerase chain reaction analysis of TF in the presence of inhibitors to these second messengers was performed in HUVECs subjected to OSS or LSS in the presence or absence of TH.
  
  
  RESULTS
  TF expression was increased and peaked at 2 hours in all HUVECs exposed to LSS or TH. Stimulation of static HUVECs with TH resulted in an increase in TF expression of 5.68 ± 1.58-, 3.80 ± 1.21-, and 2.54 ± 0.38-fold at 2, 4, and 6 hours, respectively (n = 6 experiments). In the absence of TH, HUVECs exposed to LSS demonstrated a 9.51 ± 0.62-, 7.31 ± 1.43-, and 4.39 ± 1.32-fold increase in TF expression at 2, 4, and 6 hours, respectively (n = 6 experiments). TF was increased significantly more when exposed to LSS in the presence of TH (18.85 ± 1.43-, 15.05 ± 0.95-, and 8.91 ± 1.06-fold increases at 2, 4, and 6 hours, respectively [n = 6 experiments], P < .01). Between-group analysis showed a significant difference between groups (P < .001). OSS did not significantly increase TF expression in the presence or absence of TH. ERK1/2 and p38 activation was increased in LSS and LSS + TH but not in OSS or OSS + TH (n = 3 experiments).
  
  
  CONCLUSIONS
  LSS and TH independently increased TF expression, but OSS did not. LSS + TH stimulation showed a synergistic effect, which suggests that these mechanical and chemical stimuli work through different pathways or that an intracellular interaction between TH and LSS may be present that does not occur in OSS.
authors.affiliation: Department of Vascular Surgery, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: Adrienne
authors.initials: A
authors.lastname: Rochier
authors.forename: Alexander
authors.initials: A
authors.lastname: Nixon
authors.forename: Norio
authors.initials: N
authors.lastname: Yamashita
authors.forename: Ryuzo
authors.initials: R
authors.lastname: Abe
authors.forename: Rei
authors.initials: R
authors.lastname: Abe
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Madri
authors.forename: Bauer E
authors.initials: BE
authors.lastname: Sumpio
mesh_terms: D000704:Analysis of Variance; D015153:Blotting, Western; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D040542:Mechanotransduction, Cellular / Q000187:drug effects; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D013314:Stress, Mechanical; D013917:Thrombin / Q000378:metabolism*; D013925:Thromboplastin / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D015854:Up-Regulation; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D013925:Thromboplastin; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases; D013917:Thrombin